Ferritin Associates with Interspecific Variation in Iron Accumulation among Five Economically Important Bivalves: Sequence, Expression, and Functional Analyses.

Iron is an essential micronutrient that influences both the nutritional value of bivalves and their cellular iron homeostasis. However, the molecular mechanisms underlying interspecific differences in iron accumulation among economically important bivalves remain unclear. Ferritin (FER), a highly conserved iron-storage protein, plays a central role in iron regulation, yet comparative studies integrating sequence, expression, and functional analysis across multiple bivalve species are still limited. To address this, we designed a multi-level comparative approach that integrates tissue iron measurements, sequence analyses, expression profiling, and in vitro recombinant-protein functional assays-offering a broader comparative assessment than previous single-species or single-method studies. Using this framework, we evaluated tissue iron content and FER expression characteristics in five commercially important bivalve species: Sinonovacula constricta, Meretrix meretrix, Crassostrea gigas, Ruditapes philippinarum, and Tegillarca granosa. Iron levels, measured by ICP-MS and colorimetric assays, were highest in S. constricta (approximately 2.5-fold higher than C. gigas) and lowest in C. gigas. Bioinformatic analyses, including sequence conservation, motif prediction, and structural modeling using AlphaFold, showed high conservation of FER proteins across the five species. FER mRNA expression quantified by qRT-PCR partially corresponded to tissue iron content, with the strongest agreement observed in S. constricta, which showed approximately 3-fold higher FER expression than C. gigas. Recombinant FER proteins expressed in Escherichia coli exhibited Fe